Methods and compositions for use in perfusion applications

ABSTRACT

Methods and compositions for use in perfusion applications are provided. In the subject methods, a subject or derivative thereof, e.g. isolated organ or tissue, is infused, perfused and/or transfused with at least two fluid compositions. The first fluid composition is a non-naturally occurring biological buffer free plasma-like solution and the second fluid composition is a fluid blood composition. In a preferred embodiment, an additional volume of the first solution, or a derivative thereof, is administered to the patient following introduction of the fluid blood composition. Also provided are kits and systems for performing the subject methods. The subject methods and compositions find use in variety of perfusion applications, including the treatment of hypothermic surgical applications, cryogenic procedures, and the like.

TECHNICAL FIELD

[0001] The technical field of this invention is plasma substitutesolutions and their use in perfusion applications.

BACKGROUND OF THE INVENTION

[0002] Perfusion, in which a fluid is introduced and moved through atissue or organ, e.g. via the circulatory system, plays a prominent rolein many medical applications. Such applications include treatments forblood lost during surgery or trauma, or when a tissue, organ, group oforgans or an entire subject needs to be maintained at a hypothermic orfrozen state. Such applications also include applications in which apatient's blood is flowed through an external device, such as acardiopulmonary bypass machine, where the extra circulatory volume spaceresulting from attachment of the patient's circulatory system to thedevice must be filled with a compatible blood substitute, i.e. bloodvolume expander.

[0003] Fluids that are employed in the majority of perfusionapplications are physiologically acceptable. The first physiologicallyacceptable solutions employed for perfusion applications were derivedfrom mammalian blood. Although such solutions have been used withsuccess, because such solutions are derived from natural blood, they cancontain various pathogenic substances, such as viral pathogens such asHIV, Hepatitis B, and other pathogens, e.g. prions such as thoseassociated with Cruetzfeldt-Jakob disease, and the like. Disadvantagesassociated with the use of such solutions include the need for donorsand the requirement to perform expensive screening tests to identitypathogenic agents. As such, use of blood substitute and plasmasubstitute solutions derived from natural blood are not free ofcomplication.

[0004] Accordingly, a variety of synthetic blood and plasma substitutesolutions have been developed which are prepared from non-blood derivedcomponents. Although synthetic plasma-like solutions have foundincreasing use in a variety of applications, no single solution hasproved suitable for use in all potential applications.

[0005] Therefore, there is continued interest in the development of newmethods of perfusion, as well as solutions for use therein.

[0006] Relevant Literature

[0007] Various physiologically acceptable solutions, particularly bloodsubstitute solutions, and methods for their use are described in U.S.Pat. Nos. RE 34,077; 3,677,024; 3,937,821; 4,001,401; 4,061,736;4,216,205; 4,663,166; 4,812,310; 4,908,350; 4,923,442; 4,927,806;5,082,831; 5,084,377; 5,130,230; 5,171,526; 5,210,083; 5,274,001;5,374,624; and 5,407,428.

[0008] Additional references describing physiologically acceptablesolutions, including blood substitute solutions include: Bishop et al.,Transplantation (1978) 25:235-239; Messmer et al., Characteristics,Effects and Side-Effects of Plasma Substitutes, pp 51-70; Rosenberg,Proc. 12th Congr. Int. Soc. Blood Transf.(1969); Spahn, Anesth. Analg.(1994) 78:1000-1021; Biomedical Advances In Aging (1990)(Plenum Press)Chapter 19; Wagner et al., Clin. Pharm. (1993) 12:335; ATCC Catalogue ofBacteria & Bacteriophages (1992) p 486; and 06-3874-R8-Rev. May (1987)Abbott Laboratories, North Chicago, Ill. 60064, USA.

[0009] Additional references describing various applications of suchsolutions, including hypothermic applications, include: Bailes et al.,Cryobiology (1990) 27:615-696 (pp 622-623); Belzer et al.,Transplantation (1985) 39:118-121; Collins, Transplantation Proceedings(1977) 9:1529; Fischer et al., Transplantation (1985) 39:122; Kallerhoffet al., Transplantation (1985) 39:485; Leavitt et al., FASB J. (1990)4:A963; Ross et al., Transplantation (1976) 21:498; Segall et al. FASBJ. (1991) 5:A396; Smith, Proc. Royal Soc. (1956) 145:395; Waitz et al.,FASB J. (1991) 5.

[0010] Lehninger, Biochemistry (2^(nd) Ed., 1975), pp 829ff provides areview of blood and its constituents.

SUMMARY OF THE INVENTION

[0011] Methods and compositions are provided for use in perfusionapplications. In the subject methods, a subject (e.g. an organism orderivative thereof, such as an organ or tissue) is sequentially perfusedwith at least one quantity of a plasma-like solution and at least onequantity of a fluid blood composition. In one preferred embodiment, thesubject is then perfused with at least one additional quantity of theplasma-like solution. The plasma-like solution is a non-naturallyoccurring solution that at least includes electrolytes, an oncotic agentand a dynamic buffering system. The fluid blood composition is a fluidcomposition derived from whole blood and generally comprises: red bloodcells, whole plasma or fractions thereof, whole blood, etc. Alsoprovided are kits and systems for use in performing the subject methods.The subject methods and compositions find use in a variety of differentapplications, including the treatment of hypovolemic subjects, inregional chemotherapy, in cryogenic preservation, and the like.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0012] Methods and compositions for use in the perfusion of a subjectare provided. In the subject methods, at least a plasma-like solutionand fluid blood composition are sequentially introduced into thecirculatory system of a subject. In a preferred embodiment, the subjectis then perfused with an additional volume of plasma-like solution. Theplasma-like solution is a non-naturally occurring solution comprising atleast electrolytes, an oncotic agent and a dynamic buffering system. Thefluid blood composition is whole blood or a fluid composition derivedfrom whole blood, such as purified red blood cells, whole plasma orfractions thereof. Also provided are kits and systems for carrying outthe subject methods. The subject methods and compositions find use in avariety of different applications, including the treatment ofhypovolemic subjects, regional chemotherapy, tissue and organpreservation, and the like. In further describing the subject invention,the subject solutions are detailed first followed by a discussion of thesubject methods in which the solutions find use.

[0013] Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0014] It must be noted that as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Unless definedotherwise all technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich this invention belongs.

[0015] Fluid Compositions

[0016] As mentioned above, a critical aspect of the subject invention isthe sequential administration of at least two different types of fluidcompositions: (a) a non-naturally occurring plasma-like solution; and(b) a fluid blood composition. The subject methods may further includethe administration of one or more additional types of solutions, whichsolutions are generally derivatives of the non-naturally occurringplasma-like solution. Each of the solutions finding use in the subjectinvention are described in greater detail below.

[0017] Non-Naturally Occurring Plasma-Like Solutions

[0018] The subject non-naturally occurring plasma-like solutions aresolutions that do not occur in nature, e.g. they are not produced byanimals or plants or other organisms. As such, the subject solutions aresynthetic in that they are produced through some human interaction orprocessing, such as purification, separation, genetic engineering,laboratory combination, and the like.

[0019] The plasma-like solutions of the subject invention arephysiologically acceptable, by which is meant that the solutions may beintroduced into the vasculature of a host without inherently causing atoxic reaction. The solutions have a pH ranging from about 4 to 10,usually from about 4.5 to 9 and more usually from about 5 to 8.5.

[0020] The solutions comprise a plurality-of electrolytes, including:sodium ion, chloride ion, potassium ion and calcium ion, and optionallymagnesium ion. The sodium ion concentration of the solutions ranges fromabout 70 to 160, usually from about 110 to 150, and in some embodimentsfrom 130 to 150 mM. The concentration of chloride ion in the solutionsranges from about 70 to 170, usually from about 80 to 160, more usuallyfrom about 100 to 135 and in some embodiments from about 110 to 125 mM.The concentration of potassium ion ranges from the physiological tosubphysiological, where by “physiological” is meant from about 3.5 to 5,usually from about 4 to 5 mM, and by “subphysiological” is meant fromabout 0 to 3.5, usually from about 2 to 3 mM, where in many embodimentsof the invention, the amount of potassium ion will range from about 1 to5, usually from about 2-3 mM, where in certain embodiments, the amountof potassium ion may be higher than 5 mM and range as high as about 5.5mM or higher, but will usually not exceed about 5.5 mM. The solutionsalso comprise calcium ion in an amount ranging from about 0.5 to 6.0 mM,and in many embodiments from about 0.5 to 4.0, usually from about 2.0 to2.5 mM, but in certain embodiments from about 4.0 to 6.0, usually fromabout 4.5 to 6.0 mM. Optionally, the solutions may further comprisemagnesium. When present, the magnesium ion ranges from about 0.01 to 10mM, usually from about 0.3 to 3.0 and more usually from about 0.3 to0.45 mM.

[0021] The solutions also comprise a dynamic buffering system, where theterm dynamic buffering system is used to refer to one or more reagentsthat work in combination to keep the pH of the solution in a certainrange in an in vivo environment. Preferably, the reagent members of thedynamic buffering system are normal biological components that maintainin vivo biological pH. The dynamic buffering system concept rests on thediscovery by the inventors that compounds with no intrinsic bufferingcapacity in the biological range, such as lactate, acetate, or gluconatewhich are capable of being metabolized in vivo, act with other solutioncomponents to maintain a biologically appropriate pH in an animal, evenat hypothermic temperatures and at essentially bloodless conditions. Thedynamic buffering system of the present invention depends in part onoxygenation and removal of carbon dioxide (CO₂). The dynamic buffer ofthe invention has no or substantially no ability to act as a bufferoutside of a biological system, i.e., a dynamic buffer maintains pH inthe biological range in vivo but not in a cell free environment.

[0022] A critical component of the dynamic buffering system of theinvention is a carboxylic acid, salt or ester thereof. By a carboxylicacid, salt or ester thereof is meant a compound having the generalstructural formula RCOOX, where R is an alkyl, alkenyl, or aryl,branched or straight chained, containing 1 to 30 carbons which carbonsmay be substituted, and preferably one of the carbon chains that composethe carbon chain of lactate, acetate, gluconate, citrate, pyruvate, orother biological metabolites; and X is hydrogen or sodium or otherbiologically compatible ion substituent which can associate at theoxygen position.

[0023] The solution of the present invention does not include aconventional biological buffer. By “conventional buffer” is meant acompound which in solution, in vitro, maintains pH at a particularrange. By “conventional biological buffer” is meant a compound which ina cell-free system maintains pH in the biological range of 7-8. Examplesof conventional biological buffers includeN-2-Hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid (HEPES),3-(N-Morpholino)propanesulfonic acid (MOPS),2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid(TES),3-[N-tris(Hydroxy-methyl)ethylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonicacid (EPPS), Tris[hydroxymethyl]-aminomethane (THAM), andTris[hydroxymethyl]methyl aminomethane (TRIS). Conventional biologicalbuffers have a pK in the physiological range and function mostefficiently in this range. Therefore, these buffers functionindependently of normal biological processes and are most potent incell-free systems.

[0024] The absence of a conventional biological buffer in the solutionof the invention confers several important medical advantages. Forexample, lower concentrations of buffers consisting of normal biologicalcomponents are required to maintain in vivo pH, compared to conventionalbiological buffers. Conventional biological buffers may also posetoxicity problems. Further, the absence of a biological buffer allowsthe solution to be terminally heat sterilized. Generally, medicalsolutions are preferred to be terminally heat sterilized prior to use ina patient. The term “terminally heat sterilized” or “heat sterilized” asused herein refers to the process involving heating a solution to about120° C. for 15 minutes under pressure, i.e., maintaining heat andpressure conditions for a period of time sufficient to kill all orsubstantially all bacteria and inactivate all or substantially allviruses in the solution. This procedure is normally performed in anautoclave, and is also known as “autoclaving”. The purpose of heatsterilization is to kill possible infectious agents present in thesolution. Infectious agents are known to tolerate temperatures up to100° C. It is generally considered by the art that heating a solutionunder pressure to 120° C. for about 15 minutes is sufficient to insuresterility.

[0025] The solutions also include an oncotic agent. The oncotic agent iscomposed of molecules whose size is sufficient to prevent its loss fromthe circulation by readily traversing the fenestrations of the capillarybed into the interstitial spaces of the tissues of the body. As a group,oncotic agents are exemplified by blood plasma expanders. Compoundsfinding use as oncotic agents in the subject invention may be natural orsynthetic, and will usually be polymeric compositions having an averagemolecular weight of at least about 40,000, usually at least about100,000 and more usually at least about 200,000, where oncotic agentshaving a molecular weight of 300,000 or higher may find use. Examples ofoncotic agents suitable for use in the solution of the present inventioninclude proteinaceous compounds, such as albumin, e.g. human serumalbumin, and cross-linked or high molecular weight hemoglobin,polysaccharides such as glucan polymers, and the like; organic polymers,e.g. PVP, PEG, etc.; and the like; where non-antigenic polysaccharidesare preferred.

[0026] Polysaccharides that find use as oncotic agents in the subjectsolutions include hydroxyethyl starches, hydroxymethyl alpha (1→4) or(1→6) polymers, D-glucose polymers, e.g. dextrans having an alpha (1→6)linkage, cyclodextrins, hydroxypropylstarches, hydroxyacetylstarches,and the like.

[0027] Hydroxyethyl starches are of particular interest for certainembodiments of the subject invention. The average molecular weight ofhydroxyethyl starches finding use in the subject invention may rangefrom 10,000 d to 1,000,000 d or higher, where the molecular weight willtypically range from about 40,000 d to 1,000,000 d, usually from about100,000 to 900,000, and more usually from about 200,000 to 800,000.Preferred are compositions in which the average molecular weight of thehydroxyethyl starch oncotic agent ranges from about 50,000 d to1,000,000 d, usually from about 100,000 to 900,000 and more usually fromabout 200,000 to 800,000. The degree of substitution will range fromabout 4 to 10, where in certain embodiments, the degree of substitutionwill range from 6 to 10 or 7 to 10, in other embodiments will range from4 to 6 or 4 to 5, and in other embodiments will range from 6 to 8 or 6to 7. Therefore, one class of preferred solutions will comprise ahydroxyethyl starch with between about 6 and 7 hydroxyethyl groups forevery 10 glucose units. Another class of preferred solutions willcomprise between about 4 and 6 or 4 and 5 hydroxyethyl groups for every10 glucose units. Yet another class of preferred solutions will comprisebetween about 7 and 8 hydroxyethyl groups for every 10 glucose units.

[0028] A particularly preferred oncotic agent is Hetastarch (McGaw,Inc.), an artificial colloid derived from a waxy starch composed almostentirely of amylopectin with hydroxyethyl ether groups introduced intothe alpha (1→4) linked glucose units and having a molar substitution ofabout 0.7 hydroxyethyl groups/glucose unit. The colloid properties of a6% solution (wt/wt) of Hetastarch approximates that of human serumalbumin.

[0029] Another particularly preferred oncotic agent is Pentastarch,which has a molar substitution of about 0.5 hydroxyethyl groups/glucoseunit with a range of from about 0.4 to 0.5 and an average molecularweight range (as measured by the HPSEC method as reported in PDR 1996)of from about 150,000 to 350,000 d, with 80% between 10,000 and2,000,000 d.

[0030] Another particularly preferred oncotic agent is “Hexastarch,”which has a molar substitution of about 0.6 to 0.7 (e.g. 0.64)hydroxyethylgroups/glucose unit and an average molecular weight of about220,000.

[0031] In certain embodiments, the hydroxyethyl starch will be a selectfraction of the initial hydroxyethyl starch source, particularly aselect size fraction, where generally the fraction will be a fraction inwhich substantially none of the polymeric molecules has a molecularweight greater than about 1,000,000 daltons or less than about 50,000daltons, where by substantially none is meant less than 10%, typicallyless than 5% of the polymeric molecules are above the upper threshold orbelow the lower threshold. As such the fractionated oncotic agents havereduced polydispersity. Conventional fractionation means may be used toprepare such fractions.

[0032] The concentration of oncotic agent in the solution is sufficientto achieve colloid osmotic pressure approximating that of normal humanserum, about 15 to 40 mm Hg, and in certain embodiments about 28 mm Hg.Generally, the amount of oncotic agent in the solution will range fromabout 0.5 to 30%, usually from about 1 to 25% and more usually fromabout 2 to 8%. Where the oncotic agent is a hydroxyethyl starch, theamount present in the solution will range from about 1 to 30%, usuallyfrom about 2 to 15% and more usually from about 4 to 8%.

[0033] In one aspect of the invention, the solution contains two or moreoncotic agents with differential clearance rates. The solutions of thepresent invention having two or more oncotic agents with differentialclearance rates provide additional advantages in restoring blood oncoticpressure in a hypovolemic subject over an extended period of time, whileencouraging the subject's own production of plasma proteins. Artificialoncotic agents with relatively slow clearance rates include highmolecular weight Hetastarch (molecular weight 300,000-1,000,000) anddextran 70, measured to have intravascular persistence rates of 6 hours(Messmer (1989) Bodensee Symposium on Microcirculation (Hammersen &Messmer, eds.), Karger, N.Y., pg. 59). Artificial oncotic agents withrelatively fast clearance rates include low and medium molecular weighthydroxyethyl starches with lower degrees of substitution, e.g. about0.40 to 0.65, and dextran 40, having intravascular persistence rates of2-3 hours (Messmer (1989) supra).

[0034] The solution may further comprise one or more different optionalagents which may be included in the solution to make the solution suitedfor a particular application. One optional agent that may be included,and usually is included, is sugar. The sugar will generally be a hexosesugar, such as glucose, fructose and galactose, of which glucose ispreferred. In the preferred embodiment of the invention nutritive hexosesugars are used and a mixture of sugars can be used. The sugar istypically, though not necessarily, present in the solution in aphysiological amount. By the term “physiological amount” or“physiological levels” is meant the concentration of sugar is in a rangebetween 2 mM and 50 mM with concentration of glucose of 5 mM beingpreferred. At times, it is desirable to increase the concentration ofhexose sugar in order to lower fluid retention in the tissues of asubject. Thus the range of hexose sugar may be expanded up to about 50mM or even above, but usually not above 60 and more usually not above 55mM, if necessary to prevent or limit edema in the subject undertreatment, except where the agent is present as a cryoprotective agent,as described in greater detail below.

[0035] In certain embodiments, the solutions of the present inventionmay include a blood clotting factor able to accelerate or promote theformation of a blood clot. Preferred blood clotting factors for use inthe solution of the invention include vitamin K, Factors I, II, V, VII,VIII, VIIIC, IX, X, XI, XII, XIII, protein C, von Willebrand factor,Fitzgerald factor, Fletcher factor, and a proteinase inhibitor. Theconcentration of the blood clotting factor is determined by one skilledin the art depending on the specific circumstances of treatment. Forexample, generally when vitamin K is administered, its concentrationwill be sufficient to deliver 5-10 mg to the patient.

[0036] Fluid Blood Composition

[0037] The second essential fluid composition that is employed in thesubject methods is the fluid blood composition. By fluid bloodcomposition is meant a fluid medium that is whole blood or derived fromwhole blood, such as an aqueous suspension of one or more of red bloodcells, platelets, plasma protein, albumin purified from whole blood,whole plasma or fractions thereof, e.g. fibrin free plasma, and thelike. Importantly, because of the constituent make up of the fluid bloodcomposition, the fluid blood composition is not thermally sterilizable,as such thermal sterilization irreversibly damages one or more of theconstituents of the fluid blood composition. In many embodiments, thefluid blood composition comprises one or more blood derived cellularcomponents, where cellular components includes both whole cells andfragments, portions or derivatives thereof, such as red blood cells,platelets, and the like. The preparation of such components from wholeblood is well known to those of skill in the art and any convenientmethodology for such preparation may be employed. The naturallyoccurring blood components are present in a physiologically acceptablesolution, such as the plasma-like solution described above. Fluid bloodcompositions that find use in the subject invention also include wholeblood. The blood components of the fluid blood composition may comprisedonor components or components previously harvested from the subjectundergoing the procedure in which the composition is employed. Forexample, where the fluid blood composition is whole blood, the whole maybe the patient's blood (having been previously harvested from thepatient) or donor blood. In addition, the components may besynthetically produced, e.g. recombinant albumin.

[0038] Optional Fluid Compositions

[0039] In addition to the above two fluid compositions, the subjectmethods may further employ one or more of the following optionalsolutions, where the following optional solutions are derivatives of thebasic non-naturally occurring plasma-like solution described above.

[0040] Bicarbonate Plasma-Like Solution

[0041] Bicarbonate plasma-like solutions of the subject invention aresynthetic plasma-like solutions, as described above, that furtherinclude bicarbonate ion. Any convenient source of bicarbonate ion may beincluded in the synthetic plasma-like solution in order to obtain thesubject carbonate plasma-like solution, where the bicarbonate solutionsusually include sodium bicarbonate (NaHCO₃). The concentration of NaHCO₃ranges from about 0.1 mM to 40 mM, usually from about 0.5 mM to 30 mM,and more usually from about 1 mM to 10 mM.

[0042] Bioenergetic or Supercharger Solutions

[0043] Also of interest are variations of the subject plasma-likesolutions which include elevated levels of potassium and magnesiumelectrolytes (known as “bioenergetic” or “supercharger solutions”). Byelevated levels is meant a potassium ion concentration in an amountranging from about 50 mM to 3.0 M, usually from about 200 mM to 2.5 M,and more usually from about 1.0 to 2.5 M, and a magnesium ionconcentration of from about 40 mM to 1.0 M, usually from about 0.1 to0.9 M and more usually from about 0.3 to 0.7 M. Theses solutions mayfurther comprise, in certain embodiments, bicarbonate, where thebicarbonate will be present in amounts ranging from about 0.1 to 40 mM,usually from about 0.5 to 30 mM and more usually from about 1 to 10 mM.

[0044] Oxygen Carrying Solutions

[0045] Also of interest are the subject synthetic plasma-like solutionsthat have been modified to include oxygen carrying compounds. Generally,the oxygen-carrying compound or component is present in a concentrationsufficiently low so as not to be toxic to the subject. The oxygencarrying component will usually be present in a sufficient amount todeliver enhanced oxygen to the tissues of a subject without resulting intoxicity to the subject. A “sufficient amount” of an oxygen-carryingcomponent is an amount allowing a resting subject with an unimpairedcirculation and physiology to survive and recover from trauma, illnessor injury. In normal humans at normal body temperature, this is at least5-6 ml O₂/100 ml of intravascular fluid. Oxygen-carrying componentsinclude hemoglobin extracted from human and non-human sources,recombinant hemoglobin, hemocyanin, chlorocruorin and hemerythrin, andother naturally occurring respiratory pigments extracted from naturalsources or made by recombinant DNA or in vitro methods. These compoundsmay be modified by a number of means known to the art, including bychemical crosslinking or covalent bonding to polyethylene glycolgroup(s). When the oxygen-carrying component is hemoglobin, it ispreferably present in the concentration range of between about 20-200g/l.

[0046] Instead of, or in addition to, the presence of the oxygencarrying component, the plasma-like solution may be treated in a mannerwhich increase the dissolved O₂ content of the solution to a desirablelevel, where desirable levels generally range from about 3 to 60 ml/dl,usually from about 3 to 40 ml/dl and more usually from about 4 to 25ml/dl. A variety of different technologies are available for oxygenatingsolutions, where such technologies include: pressurizing with hyperbaricoxygen; bubble oxygenation; passing the solution through an oxygenatingmembrane, such as the those sold by Terumo Corporation (Japan); and thelike.

[0047] Cryogenic Solutions

[0048] Cryogenic solutions are also provided by the subject invention.Any of the above synthetic plasma-like solutions may be modified toinclude one or more cryoprotective agents to produce a cryogenicsolution of the invention, where by cryoprotective agent is meant anyagent that preserves the structural integrity of tissue underhypothermic, e.g. sub-zero, conditions, where in certain embodiments thecryoprotective agent will be an agent that modulates or influences, atleast to a partial extent, the ordered crystal arrangement of watermolecules. Cryoprotective agents of interest include: alcohols,particularly low molecular weight aliphatic alcohols, usually C₁ to C₆alcohols, more usually C₁ to C₄ alcohols, such as methanol, ethanol, andthe like; polyols, including linear, branched and cyclic polyols,usually low molecular weight aliphatic polyols, including diols, triols,and other polyols, such as sugars (described in greater detail below)where polyols of particular interest include diols, such asethylenediol, propanediol, butanediol, triols, e.g. glycerol, and thelike; sugars, including erythrose, threose, ribose, arabinose, xylose,lyxose, allose, atrose, glucose, mannose, gulose, idose, galactose,talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose,tagatose and disaccharides, e.g. sucrose, lactose and maltose, whereglucose is particularly preferred; other agents such as timethylamine,trimethylamine oxide (TMAO), DMSO, urea, formamide, dimethylformamideand the like; clathrates, silicon comprising agents, such as silanes andthe like, fluorocarbon compounds and derivatives thereof; etc; where thecryoprotective agent may be forced into solution by pressure and/or asuitable surfactant agent may be employed, where such surfactant agentsare known to those of skill in the art. Such agents will typically bepresent in amounts sufficient to provide the desired cryoprotectiveeffect, where the particular amount of the agent will depend on theparticular agent employed. When the agent is a polyol, e.g. a diol, itwill generally be present in amounts ranging from about 0.2 to 1 M or 0to 30%. With respect to propanediol, in particular a range of 0.2 M to0.6 M is preferred and a concentration of about 0.4 M propanediol ismost preferred. 1,2 propanediol is preferred as the adduct to thesolution used for low temperature organ and donor preservation accordingto the invention, although 1,3 propanediol may be used. For TMAO, TMAOwill be present in the solution in a final concentration in a rangebetween 0.2 M and 7 M. When glycerol is employed, it will be present ina concentration ranging from about 0 to 40%, usually from about 5 to30%, and more usually 5 to 20%. When DMSO is employed, it will bepresent in amounts ranging from about 0 to 40%, usually from about 5 to30%, and more usually from about 5 to 20%. When a sugar is employed(particularly glucose), the sugar ranges between about 0.6 M to about1.4 M, with 1.0 M being preferred for certain embodiments.

[0049] Methods of Preparing the Fluid Compositions

[0050] In preparing the subject solutions and fluid compositions, thevarious constituents may be combined at substantially the same time, oradded sequentially, as may be convenient. In most situations, thenon-naturally occurring plasma-like solutions may be terminally heatsterilized as described above. As also described above, the solutionsmay further comprise agents that should not be terminally heatsterilized, such as a source of bicarbonate, where the bicarbonateparticipates in the dynamic buffering system. In such instances, thesodium bicarbonate will be added as a sterile solution to apre-autoclaved “base solution.” Similarly, when it is desirable to add ablood clotting factor or oxygen-carrying component, the blood clottingfactor or oxygen-carrying component is added as a sterile solution tothe autoclaved base solution.

[0051] For purposes of description of the invention, the mixtureaccording to the invention has been discussed and will continue to bediscussed in terms of an aqueous solution. From the followingdescription of the invention, it is expected that one of ordinary skillin the art would be enabled to provide the mixture as a dry mixturewhich can then be later hydrated.

[0052] Methods of Use

[0053] The above described solutions find use in methods of perfusing asubject. The term “subject” is used broadly to refer to any biologicalentity having a circulatory system, and therefore includes: wholeorganisms, e.g. mammals, including dogs, cats, rodents, cows, horses,and humans; as well as derivatives of such organisms, such as a tissuesor organs, e.g. the heart, liver, kidney, etc.

[0054] In practicing the subject methods, at least two fluidcompositions are sequentially administered to the subject, or derivativethereof. The first fluid composition is the plasma-like solution,described above. The second fluid composition is the fluid bloodcomposition. By sequentially administered is meant that the first fluidcomposition is administered to the subject prior to the second fluidcomposition. The subject invention also encompasses those methods inwhich two or more quantities of the first fluid composition areadministered and/or two or more quantities of the second type of fluidcomposition are administered. Thus, in the broadest sense the subjectmethods are directed to methods in which at least one administration ofthe first type of fluid composition is administered followed by at leastone administration of the second type of fluid composition.

[0055] In many embodiments, one or more additional fluid compositions,e.g. the bicarbonate plasma-like solution, the cryogenic solution, thesupercharger solution etc., are administered to the host. Theseadditional fluid compositions may be administered at any time prior to,between or after the first and second fluid compositions, depending onthe particular method being performed. In certain embodiments, thefollowing sequential order of fluid compositions is administered to thesubject: (1) plasma-like solution; (2) bicarbonate plasma-like solution;(3) bioenergetic solution; (4) bicarbonate plasma-like solution; and (5)fluid blood composition. In yet other embodiments, the following orderof fluid compositions is administered to the subject: (1) plasma-likesolution; (2) bicarbonate plasma-like solution; (3) bioenergeticsolution; (4) cryogenic solution; (5) bicarbonate plasma-like solution;and (6) fluid blood composition.

[0056] In a class of preferred embodiments, administration of the fluidblood composition is followed by administration of an additional volumeof the non-naturally occurring plasma-like solution or a derivativethereof, e.g. the plasma-like solution; the bicarbonate plasma-likesolution; the bioenergetic solution; the cryogenic solution; etc.

[0057] In general, the fluid compositions according to the invention areadministered using an intravenous line using a gravity feed line or apumped circulating device such as a centrifugal pump, roller pump,peristaltic pump or other known and available circulatory pump. Whenemployed, the circulating device is connected to the subject viacannulae inserted surgically into appropriate veins and arteries. Forexample, when the solution is administered to a chilled subject, it isgenerally administered via an arterial cannula and removed from thesubject via a venous cannula and discarded, stored or circulated.

[0058] Depending on the particular method being performed and conditionbeing treated, the subject methods may further include a step in whichthe temperature of the subject is modulated, e.g. raised or lowered fromambient temperature. In many embodiments, the temperature of the subjectis lowered for at least a portion of the treatment process, i.e. theprocess in which the at least two fluid compositions are administered tothe subject. Where the temperature of the subject is lowered, thetemperature will generally be lowered to at least about 32, usually atleast about 20 and more usually at least about 5° C., where thetemperature may be lowered to −80° C. or lower, but will generally notbe lowered to below −196° C. The temperature of the subject may bemodulated using any convenient protocol, such as the use of temperaturecontrolled rooms, warming or cooling blankets, perfusion with chilled orwarmed solutions, immersion in cooling or warming fluids, etc.

[0059] Alternatively or in addition, the pressure of the subject may bemodulated. As such, the subject may be pressurized, i.e. placed in ahyperbaric environment, for at least a portion of the subject methods.In the hyperbaric environment, the pressure is typically at least about1.5 atm, and usually at least about 2.0 atm, where the pressure may beas high as 200 atm or higher, but will generally not exceed about 10,000atm, usually will not exceed about 5000 atm more usually will not exceedabout 2500 atm. The hyperbaric environment may be provided using anyconvenient technology. See e.g. U.S. Pat. Nos. 5,738,093; 5,678,722;5,678,543; 5,398,678; 5,109,837; 5,060,644; 4,974,829; 4,837,390;4,727,870; 4,655,048; 4,633,859; the disclosures of which are hereinincorporated by reference. For example, a thick walled chamberpressurized with a gaseous medium, e.g. helium, argon, krypton, neon,may be employed to provide the hyperbaric environment.

[0060] The subject methods in which a subject is sequentially perfusedwith at least a synthetic plasma-like solution and a fluid bloodcomposition find use in a variety of different applications, where suchapplications include: hypothermic surgical applications, hyperbaricsurgical applications, organ or organism preservation, and the like. Thefollowing applications are representative of applications in which thesubject methods find use.

[0061] One type of application in which the subject methods find use ishypothermic surgical applications, such as hypothermic cardiac surgeryin which a cardiopulmonary bypass device is employed. In such methods,the subject is prepared for surgery in accordance with standardprocedures. The subject is connected to the cardiopulmonary bypassmachine according to the accepted protocol which depends on theparticular machine being employed. A variety of differentcardiopulmonary bypass machines, as well as protocols for their use, areknown to those of skill in the art and include those described in U.S.Pat. Nos. 5,827,220; 5,820,579; 5,800,375; 5,785,686; 5,688,245;5,643,921; 5,478,309; 5,437,601; 5,383,854; 5,383,839; 5,334,136;5,308,320; 5,300,015; 5,254,097; 5,158,539; 5,011,469; 4,808,163;4,804,365; 4,690,002; 4,553,532; 4,398,872; 4,293,961; the disclosuresof which are herein incorporated by reference. In preparing for surgery,a quantity of the synthetic plasma-like solution of the invention isadministered to the subject as needed to prevent and/or treathypovolemia during the preparation process, e.g. the instrumentation andcannulation of the subject, connection of the subject to thecardiopulmonary bypass device, etc. Generally, the amount of plasma-likesolution that is administered ranges from about 0.25 l to 101 l, usuallyfrom about 0.50 to 5.0 l and more usually from about 1.0 to 3.0 l.

[0062] Following preparation of the patient and completion of thecircuit with the cardiopulmonary bypass, the sodium bicarbonate solution(as described above) is introduced into the circuit in a mannersufficient to replace substantially all of the patients blood from thepatient's circulatory system. Substantially all of the patient's bloodis considered to have been removed from the patient when the hematocritof the patient falls below about 15%, usually below about 7% and moreusually below about 3%.

[0063] Once substantially all of the patient's blood has been replacedwith the bicarbonate priming solution, the temperature of the patient iscooled and a quantity of concentrated KCl solution is introduced intothe circuit in a manner sufficient to achieve cardiac arrest. Thesubject or patient is cooled to a temperature of from about 28 to 1° C.,usually from about 8 to 2° C. The concentrated KCl solution has aconcentration sufficient to increase the potassium ion concentration inthe fluid present in the subject's circulatory system to a value rangingfrom about 5 to 300 mM, usually from about 6 to 50 mM. As such, theconcentration of the KCl solution ranges from about 0.3 to 3M, usuallyfrom about 0.5 to 2.8 M and more usually from about 1.5 to 2.5 M. TheKCl solution may be made up of solely KCl and purified water, or maycomprise one or more additional components, such as magnesium,bicarbonate, lactate, and the like. The amount of concentrated KClsolution that is introduced into the circuit is generally at least about2 ml, usually at least about 10 ml and more usually at least about 50ml, where the amount may be as high as 400 ml or higher, but willtypically not exceed about 1 l and usually will not exceed about 500 ml.

[0064] Following introduction of the concentrated KCl solution, thetemperature of the host or subject is lowered to the temperature desiredfor surgery, e.g. to a temperature between about 30 and 0° C., usuallybetween about 25 and 1° C. and more usually between about 10 and 2° C.,often by the addition of one or more liters of bicarbonate solution.Just prior to circulatory arrest surgery (i.e. where the heart hasstopped beating), the bioenergetic solution is introduced. The amount ofbioenergetic solution introduced is at least about 1 ml, usually atleast about 100 ml, and more usually at least about 500 ml, where theamount may be as high as 4 l or higher, but will typically not exceedabout 3 l and usually will not exceed about 2 l.

[0065] The above steps result in a subject under cardiac arrest andprofound hypothermia, where the temperature of the subject ranges fromabout 30 to 2° C., usually from about 10 to 2° C. These conditions aremaintained during the particular hypothermic surgical procedure beingperformed.

[0066] Following completion of the surgical procedure, the circuit isflushed with fresh biocarbonate plasma-like solution and the subject isgradually warmed to a temperature ranging from about 0 to 20° C.,usually from about 2 to 12° C. One or more additional flushes withbicarbonate solution may be employed. Once the temperature of thesubject reaches 4 to 28° C., the fluid blood composition, e.g. wholeblood, is introduced into the circuit in a manner sufficient to raisethe hematocrit to where cardiac function may be restored, where cardiacfunction may be restored via mechanical means, electrical means,pharmaceutical means, spontaneous defibrillation, and the like, as isknown to those of skill in the art. For example, whole blood is infuseduntil the subject achieves an acceptable hematocrit, generally exceedinghematocrits of about 28%. When an acceptable hematocrit is achieved andperfusion is discontinued, the subject is revived after closure ofsurgical wounds using conventional procedures.

[0067] The subject methods also find use in cryogenic preservationapplications, in which an organism or derivative thereof, either livingor non-living, is to be preserved for an extended period of time. Insuch methods, the subject or derivative thereof is first cooled to ahypothermic temperature ranging from about 35 to 0° C. usually fromabout 30 to 5° C. and more usually from about 10 to 0° C., where incertain embodiments the hypothermic temperature ranges from about 20 to0° C. and usually 12 to 0° C., using any convenient protocol, e.g.cooling blankets, perfusion with cooled fluids, etc. During this initialcooling process, the subject is perfused with the synthetic plasma likesolution as described above, such that substantially all of thesubject's blood is removed and replaced with the synthetic-plasma likesolution.

[0068] Following cooling of the subject and replacement of the subject'sblood with the synthetic plasma-like solution, which may or may not bethe bicarbonate plasma-like solution, the hypothermic subject is movedto a hyperbaric environment, e.g. a walled chamber of sufficientstrength to withstand the pressures to be produced inside the chamber.For example, a thick-walled chamber made of a suitably strong materialand appropriate construction, e.g. cryogenic steels and the like, may beemployed. The chamber may be equipped with heating and cooling means, aswell as means for monitoring the subject and means for introducing andremoving fluids from the circulatory system of the subject present inthe chamber. The subject is pressurized by introducing a sufficientquantity of a suitable gas into the chamber. A variety of gases may beemployed, including helium, as well as other noble gases, such as argon,krypton, or neon. The subject is then pressurized to the desiredhyperbaric pressure, which generally ranges from about 2 to 10,000 atm,usually from about 100 to 5000 atm and more usually from about 300 to3000 atm.

[0069] Of particular interest is the subjection of the patient toconditions sufficient for ICE3 formation. Under such conditions, thetemperature of the subject is reduced to a value ranging from about −1to −200, usually from about −5 to −40 and more usually from about −15 to−25° C. The pressure of the subject typically ranges from about 10 to5000, usually from about 500 to 3000 atm.

[0070] In certain embodiments of the above preservation procedures, itis desirable to introduce a cryogenic solution (as described above) intothe subject. Where a cryogenic solution is employed, the temperature towhich the subject is lowered for storage may range from about −2 to−270° C., usually from about −40 to −250° C. and more usually from about−60 to −200° C.

[0071] Using the above protocols, the subject may be stored forindefinite periods of time. Generally, the subject will be maintained ina pressurized and hypothermic state during storage. However, in certainembodiments, it may be possible to depressurize the subject where thesubject is preserved in a metastable state.

[0072] Following storage of the subject, the subject may be graduallywarmed and depressurized in a manner that provides for minimal tissuedamage. At some point during this process, generally when thetemperature is between about 8 and 28° C. and the pressure is betweenabout 1 and 5 atm, the subject is perfused with the fluid bloodcomposition, e.g. whole blood. The subject may or may not be revived.

[0073] Kits

[0074] Also provided by the subject invention are kits for use inperforming the subject methods. The subject kits at least include thesynthetic plasma-like solution in combination with instructions forpracticing the subject methods. The amount of synthetic plasma-likesolution that is included in the kits may vary, but will generally rangefrom about 0.5 to 1000 l, usually from about 1 to 300 l and more usuallyfrom about 1 to 100 l. The solution may be present in any convenientcontainer or package, such as a flexible polymeric bag, and the like.The instructions for practicing the subject method may be present on oneor more of a package insert, the containing or packaging of the device.The subject kits may further comprise one or more additional fluidcompositions, depending on the particular method to be performed. Theseadditional fluid compositions include: a concentrated KCl solution, abicarbonate solution, a bioenergetic solution, a heta freeze solutionand the like, where the kits may include one or more of these varioussolutions pre-made or may include components that can be combined at thetime of use with the provided synthetic plasma-like solution in order toprepare the solution of interest. In addition, the subject kits mayfurther include the fluid blood composition that is employed in themethods, particularly where the subject's own blood is to be discardedand replaced with donor blood or blood components.

[0075] Systems

[0076] Also provided are systems for use in performing the subjectmethods. The subject systems are specifically designed to optimallysequentially deliver the plasma-like solution and fluid bloodcomposition to a subject under controlled temperature and pressureconditions. As such, the subject systems may include: circuitry forestablishing fluid communication with the circulatory system of thehost; oxygenators; pumps to move the subject fluid compositions throughthe circulatory system of the host; heat exchangers, dialysis circuits;computers and monitors to collect, store, process and display data, e.g.pressure and temperature; means for establishing a hyperbaricenvironment, e.g. a tent or chamber for delivering hyperbaric oxygen tothe subject; means for controlling the temperature of the subject, e.g.warming and cooling blankets; and the like.

[0077] The following examples are offered by way of illustration and notby way of limitation. EXPERIMENTAL I. Fluid Compositions: A. Plasma-LikeSolution 1 to 10% High Molecular Weight Hetastarch (average molecularwt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++ 0-10 mM lactate 1-40mM glucose 0-50 mM B. Bicarbonate Plasma-Like Solution 1 to 10% HighMolecular Weight Hetastarch (average molecular wt. of 350,000-900,000)Ca++ 1-6 mM K+ 1-5 mM Mg++ 0-10 mM lactate 1-40 mM glucose 0-50 mMbicarbonate 5-10 mM C. Bioenergetic Solution (please providecomposition) K+ 100 to 3000 mM Mg++ 30 to 1000 mM D. CryoprotectiveSolutions 1. High Molecular Weight Hetastarch 1 to 10% (averagemolecular wt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++ 0-10 mMlactate 1-40 mM glucose 0-50 mM bicarbonate 5-10 mM glycerol 10-20%  2.High Molecular Weight Hetastarch 1 to 10% (average molecular wt. of350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++ 0-10 mM lactate 1-40 mMbicarbonate 5-10 mM glycerol 10-20%  3. High Molecular Weight Hetastarch1 to 10% (average molecular wt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5mM Mg++ 0-10 mM lactate 1-40 mM glucose 0-50 mM bicarbonate 5-10 mMglycerol 5-15% DMSO 5-15%

[0078] II. Hypothermic Cardiovascular Surgery

[0079] The patient is anesthetized, instrumented and catheterized. Aportion of the patient's blood volume (I liter), is collected andreplaced with a liter of a Plasma-Like Solution. The patient's chest isthen opened. The volume of blood lost in these procedures is replacedfirst with collected blood, and then with Plasma-Like Solution. Theaorta and the vena cava are cannulated for cardiopulmonary bypass. Thepatient's circulation is connected to the bypass circuit (filled with aBicarbonate Plasma-Like Solution) which contains a blood pump, anoxygenator and a heat exchanger. The patient's body temperature is thenlowered to 14° C. and the circulating blood is replaced with theBicarbonate Plasma-Like Solution. This is continued until the hematocritapproaches 1%. During this blood volume replacement, the remainder ofthe patient's blood is collected for use during warming, followed by theintra-arterial injection of 100 ml of a 2 mEq/ml KCl solution to arrestcardiac fibrillation. After virtually all the patient's blood isreplaced with a Bicarbonate Plasma-Like Solution, the body temperatureis lowered to approximately 4° C., and 500 ml of the BioenergeticSolution (375 ml of a 2 mEq/ml KCl solution and 125 ml of a 50%MgSO₄.7H₂O solution) is introduced intra-arterially and circulated for 4minutes.

[0080] Following the completion of surgery, the circuit is cleared ofthe Bicarbonate Plasma-Like Solution to which was added the BioenergeticSolution. The patient is then warmed, and flushing is then continuedwith enough Bicarbonate Plasma-Like Solution to lower the blood plasmaK+ concentration to below 6 mEq/L. At 14° C., diluted blood initiallycollected from the patient is returned, and as the patient becomeswarmer, more blood is added. When the patient is removed from bypass,the remainder of the blood cells in the circuit is returned to thepatient, as well as any of the patient's remaining whole blood collectedat the beginning of the procedure. The patient is then warmed to normalbody temperature, all incisions are closed, and the patient maintainedon anesthetics and a ventilator until awakened the next day.

[0081] III. Cryogenic Preservation

[0082] A 50 g hamster is injected with ketamine and placed in crushedice. When its body temperature sinks to 12° C., the hamster is thenplaced underneath a stereo microscope, instrumented, cannulated,ventilated with 100% O₂, chilled to 1° C. Its blood is replaced with 4ml of the Bicarbonate Plasma-Like Solution, then 4 ml of theBioenergetic Solution and then with 10 ml Cryoprotective Solution 1. Thehamster is placed in a thick walled chamber and compressed to 1500 atmof helium, while its temperature is lowered stepwise to −20° C. Thetemperature is then lowered to −196° C., and the hamster slowlydepressurized. After storage at this temperature for 1 week, the hamsteris re-pressurized to 1500 atm with helium. It is then warmed to −20° C.,then slowly depressurized while warming to 1° C. The hamster is againperfused with the Bicarbonate Plasma-Like Solution, then warmed whilebeing perfused with whole blood. The animal is ventilated with 100% O₂,brought to normal temperature.

[0083] IV. 59 patients undergoing major elective surgery forgastrointestinal, urological, gyecological and orthopedic pathology wereintraoperatively infused with intraveneous Plasma Like Solution inresponse to loss in blood pressure. Of these patients, 31 lostsufficient amounts of blood to require transfusion with packed red bloodcells following infusion with the Plasma-Like Solution. The averageamount of Plasma-Like Solution that was infused into these patients wasapproximately 2.1 l. The average amount of packed red blood cells thatwas transfused was approximately 1 l. In a number of patients,additional Plasma-Like Solution was then infused, followed bytransfusion of an additional amount of packed red blood cells. Allpatients successfully recovered from their operations and there were noserious related adverse events related to the use of the Plasma LikeSolution.

[0084] It is evident from the above results and discussion that thesubject invention provides for methods of perfusion of a subject thatfind use in a variety of different applications. Use of the subjectmethods results in better outcomes in a variety of differentapplications, including hypothermic surgery, cryogenic preservation, andthe like.

[0085] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0086] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method of perfusing a subject, said methodcomprising: sequentially introducing to said subject at least onequantity of first, second and third types of fluid compositions,wherein: (a) said first fluid composition is a conventional biologicalbuffer free non-naturally occurring plasma-like solution comprising: (i)electrolytes; (ii) an oncotic agent; (iii) and a dynamic bufferingsystem; (b) said second fluid composition is a fluid blood composition;and (c) said third fluid composition is the same as said first fluidcomposition or is a derivative thereof.
 2. The method according to claim1, wherein said electrolytes include: Na⁺, Mg²⁺, Ca²⁺, and Cl⁻.
 3. Themethod according to claim 2, wherein said non-naturally occurringplasma-like solution further includes K⁺.
 4. The method according toclaim 1, wherein said oncotic agent is a polymer.
 5. The methodaccording to claim 1, wherein said dynamic buffering system comprises anorganic carboxylic acid, salt or ester thereof.
 6. The method accordingto claim 1, wherein said non-naturally occurring plasma-like solutionfurther includes a sugar selected from the group consisting ofmonosaccharides and disaccharides.
 7. The method according to claim 1,wherein said fluid blood composition is whole blood or a derivativethereof.
 8. The method according to claim 7, wherein said fluid bloodcomposition is not thermally sterilizable.
 9. The method according toclaim 1, wherein said method further comprises the introduction of atleast one additional fluid composition.
 10. The method according toclaim 1, wherein said method further comprises reducing the temperatureof said subject.
 11. The method according to claim 1, wherein saidmethod further comprises placing said subject into a hyperbaricenvironment.
 12. A kit for use in a perfusion method, said kitcomprising: (a) a conventional biological buffer free non-naturallyoccurring plasma-like solution comprising: (i) electrolytes; (ii) anoncotic agent; (iii) and a dynamic buffering system; and (b)instructions for performing the method according to claim
 1. 13. The kitaccording to claim 12, wherein said electrolytes include: Na⁺, Mg²⁺,Ca²⁺, and Cl⁻.
 14. The kit according to claim 13, wherein saidnon-naturally occurring plasma-like solution further includes K⁺. 15.The kit according to claim 12, wherein said oncotic agent is a polymer.16. The kit according to claim 15, wherein said polymer is apolysaccharide.
 17. The kit according to claim 16, wherein saidpolysaccharide is a starch.
 18. The kit according to claim 12, whereinsaid dynamic buffering system comprises an organic carboxylic acid, saltor ester thereof.
 19. The kit according to claim 12, wherein saidnon-naturally occurring plasma-like solution further includes a sugarselected from the group consisting of monosaccharides and disaccharides.20. The kit according to claim 19, wherein said sugar is glucose. 21.The kit according to claim 20, wherein said kit further comprises atleast one additional fluid composition.
 22. The kit according to claim21, wherein said additional fluid composition is a fluid bloodcomposition.
 23. The method according to claim 22, wherein said fluidblood composition is whole blood.
 24. A perfusion system comprising:means for introducing and removing liquid from the circulatory system ofa subject; (a) a conventional biological buffer free syntheticplasma-like solution comprising: (i) electrolytes; (ii) an oncoticagent; (iii) and a dynamic buffering system; and (b) a fluid bloodcomposition.
 25. The system according to claim 24, wherein said systemfurther comprises a means for modulating the temperature of saidsubject.
 26. The system according to claim 24, wherein said systemfurther comprises a means for modulating the pressure of the environmentof said subject.